Biolog Phenotypic Profiling of Dinoroseobacter shibae

D. shibae Dshi-5 and Dshi-6 strains were tested for their ability to utilize 95 different carbon sources using the Biolog PM01 microplate (34 (link)). Biolog experiments were performed with freshly grown D. shibae cells that were resuspended in 10 mL inoculating fluid IF-0 to achieve 85% transmittance in the Biolog turbidimeter and with a negative control without bacteria. A solution containing 428 μL H₂O, 120 μL dye D, 1,200 μL 10× seawater medium (160 g NaCl, 32 g NaSO₄, 24 g MgCl₂ · 6H₂O, 4 g KCl, 2 g NH₄Cl, 1.6 g KH₂PO₄, and 1.2 g CaCl₂ · 2H₂O per L), 120 μL 100× NaHCO₃ buffer (1.9 g/100 mL), 120 μL 100× vitamin mix (2 mg biotin [B₇], 20 mg niacin [B₃], 8 mg 4-aminobenzoic acid [B₁₀], and 1 mg thiamine [B₁] per L), and 12 μL 1,000× trace element solution (2.1 g FeSO₄ · 7H₂O, 13 mL 25% HCl, 5.2 g Na₂EDTA, 30 mg H₃BO₃, 100 mg MnCl₂ · 4H₂O, 190 mg CoCl₂ · 6H₂O, 24 mg NiCl₂ · 6H₂O, 2 mg CuCl₂ · 2H₂O, 144 mg ZnSO₄ · 7H₂O, and 36 mg Na₂MoO₄ · 2H₂O per liter) was added to a final volume of 12 mL. Each well of the PM01 plate was inoculated with the cell suspension, and the microplates were incubated at 28°C for 96 h in the OmniLog plate reader. The OmniLog data were processed and analyzed using the R package opm v1.1.0 (70 (link)). Curve parameters were estimated using spline fitting, and the curve maximum was used for plotting the heatmap and comparing the strains.